CN111088283B

CN111088283B - mVSV viral vector, viral vector vaccine thereof and mVSV-mediated novel coronary pneumonia vaccine

Info

Publication number: CN111088283B
Application number: CN202010203073.6A
Authority: CN
Inventors: 秦晓峰; 吴飞; 韦治明; 权海峰; 龙丽梅
Original assignee: Suzhou Ultralmmune Co ltd
Current assignee: Ruifengkang Biomedical Technology Zhejiang Co ltd
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-06-23
Anticipated expiration: 2040-03-20
Also published as: WO2021185310A1; CN111088283A

Abstract

mVSV virus vector, namely attenuated mVSV obtained after multiple modification mutations occur to M protein amino acid site of wild Indiana strain VSV, and simultaneously preferred heterologous antigen gene is preferentially integrated to double cloning site region of pmVSV-Core of mVSV packaging Core plasmid; a mVSV viral vector vaccine comprising a heterologous antigen gene of a target virus fused or chimeric in between mVSV vector envelope G and L genes, the antigen gene comprising a chimeric antigen gene, a chimeric combination antigen gene or a fused antigen gene encoding the target virus envelope; the mVSV viral vector is embedded or fused with the dominant antigen of the spike protein S of the COVID-19 viral pathogen, and the dominant antigen is preferably selected from the receptor binding domain of the spike protein S, namely RBD, so that a mVSV-mediated-based new coronary pneumonia vaccine is formed, and the vaccine has better prevention or treatment effect on a new coronary pneumonia virus infected person.

Description

mVSV viral vector, viral vector vaccine thereof and mVSV-mediated novel coronary pneumonia vaccine

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an mVSV viral vector, a viral vector vaccine thereof and a new vaccine for coronary pneumonia based on mVSV mediation.

Background

Coronaviruses (Coronavirus) belong to members of the order nidovirales (order Nidovirals), the family coronaviridae (family famearly) and the genus coronaviruses (genus Coronavirus) in virological classification, have single-stranded and positive-stranded RNA genomes, have the total genome length of 26-32 kb, and are RNA viruses with the largest genome known at present. Coronavirus is widely infected in nature, and common mammals such as dogs, cats, mice, pigs, cattle and poultry are susceptible to the coronavirus. In recent years, various types of coronaviruses have been isolated from whales and camels, especially bats. Six human coronaviruses are currently known, namely human coronaviruses 229E (HCoV-229E) and HCoV-OC43 found in the 60 s of the twentieth century, SARS-CoV appearing in 2003, HCoV-NL63 isolated in the netherlands in 2004, HCoV-HKU1 identified in hong kong in 2005, and the novel Middle East Respiratory Syndrome (MERS) coronaviruses MERS, appearing in the Middle East of 2012. The COVID-19 virus has high homology with the gene of SARS (Severe acid respiratory syndrome). In the process of infecting host cells by 2019-nCoV, firstly, a spike protein (S protein) of the virus recognizes a receptor protein ACE2 (angiotensin-converting enzyme 2) on cell membranes, and then mediates and promotes the fusion of virus envelopes and the cell membranes, so that the virus finally invades the host cells. Coronaviruses can infect the respiratory tract, digestive tract, liver, kidney and nervous system of the body, causing pathological injuries of various degrees, and even death of serious people.

Coronaviruses in terms of phylogenetic analysis of nucleic acid sequences, the International Commission on virology (ICTV, 2012) in the ninth report divided the members of the genus coronaviruses into α, β, γ and δ groups into four groups.human coronaviruses were distributed mainly in α and β groups.A HCoV-229E and HCoV-NL63 were located in α, HCoV-OC43 and HCoV-HKU1 were located in subgroup 2a in β, MERS-CoV was in subgroup 2c in β, while the most recent globally used COVID-19 virus and SARS was in subgroup 2b in β.

Since the outbreak of the pneumonia epidemic situation of the novel coronavirus (COVID-19), more than 8 million people are infected in the whole country and nearly 4000 people die by the current outbreak; and has caused more than 50000 people to infect on the global scale, thousands of people die, and is a coronavirus with extremely strong transmission. The COVID-19 virus coronavirus is different from the traditional coronavirus in that the coronavirus is susceptible to all people, and can infect the upper respiratory tract to cause common cold symptoms such as fever, cough, laryngitis and the like, and can infect the lower respiratory tract to cause acute respiratory symptoms such as bronchitis, pneumonia and the like. Recent studies have shown that the virus has 10 times more ability to bind ACE2 in the S spike protein RBD segment than SARS, and is most transmissible among the seven currently known human-infecting coronaviruses. The COVID-19 virus is similar to SARS coronavirus in structure, is an enveloped single-stranded positive-strand RNA virus, and the spinous process protein S protein embodied on the surface of the virus is a specific tissue structure on the virus envelope, and a large number of spike proteins are formed on the surface of the virus, thereby playing an important role in the invasion of the virus into target cells and the recognition of the virus and the cells. Many studies have shown that the S protein vaccine of SARS can generate high titer neutralizing antibody against SARS-CoV virus, and can effectively prevent SARS-CoV infection, so the S antigen of new corona is usually used as main target when developing new coronavirus vaccine, considering that the three-dimensional structure of COVID-19 virus is highly similar to that of SARS S protein.

The global wide outbreak of new coronary disease causes that the development of medicines for the new coronary pneumonia is reluctant, the research and report results of JAMA (national institute of medical science) of American medical society indicate that part of recovered patients are still carriers of the new coronary virus, and further the discussion of whether the new coronary virus becomes a global epidemic disease is initiated, the new coronary virus may be popular in human society for a long time like influenza virus, and the vaccine is proved to be the most cost-effective, most effective and most lasting disease prevention and control measure, so that the inoculation of the new coronary vaccine for the whole people is imperative, and the research and the release of the new coronary pneumonia vaccine in a short time are the most powerful means for preventing the spread of the epidemic situation.

Research shows that pathogens causing serious infectious diseases such as Human Immunodeficiency Virus (HIV), influenza virus, severe acute respiratory syndrome virus (SARS-CoV) and the like invade and infect organisms through mucosal surfaces (genital tract, respiratory tract and gastrointestinal tract), and because the organisms cannot induce effective mucosal immune response to eliminate mucosal infection pathogens, the pathogens rapidly diffuse into blood and further invade the whole body, so that the organisms, particularly lung tissues, are damaged.

It is known that conventional vaccines such as inactivated, protein vaccines, DNA vaccines, subunit vaccines, etc., often do not induce specific mucosal immune responses by conventional routes of immunization (intramuscular injection, subcutaneous, etc.). Regardless of the form of the vaccine, to induce a mucosal immune response usually requires vaccination of the target antigen from a mucosal site before it can be taken up and presented by APCs in mucosal tissues, further activating the mucosal immune system, inducing a strong mucosal immune response.

The bottleneck of the existing mucosal vaccination with traditional antigens is: frequent physical agitation of local cilia of the mucosa can rapidly remove foreign antigens; a large amount of acidic solution exists on local mucosa, is rich in hydrolase, DNase and the like, and can quickly degrade foreign antigens. Therefore, conventional antigens administered to mucosal sites, which are rapidly eliminated and degraded, do not stay locally and effectively on the mucosa, are not sufficiently presented by APCs, and do not induce an effective mucosal immune response. Even if induced, the extent of response was very low.

Known vaccine vectors, wild strains of Vesicular Stomatitis Virus (VSV), can infect a wide variety of animals and insects in their natural environment. The livestock is naturally infected with VSV, such as horses, cattle (sheep) and pigs, but the human population hardly has active infection of vesicular stomatitis virus in the natural state, and the infection of human does not cause obvious diseases, so that the Vesicular Stomatitis Virus (VSV) serving as a virus vector vaccine has natural advantages compared with other vectors, so that the VSV serving as a virus vector chimeric or fused target antigen can enhance the immune response strength of the organism, the VSV virus vector vaccine further adopts an immune mode of mucosal part inoculation to induce the organism to generate stronger specific mucosal immune response, when foreign pathogens invade through the mucosa, mucosal tissues can be activated to rapidly eliminate the pathogens, further the VSV virus vector is known to have the characteristics not possessed by other tool vectors, and when the designed preventive vaccine is used for preventing enveloped viruses, the VSV can completely display the envelope protein of the target virus on the envelope protein GP of the VSV, antigen protein is fully exposed on the surface of the recombinant virus, the virus vector vaccine still has specific immune response for effectively activating an organism after in vitro inactivation, the immunogenicity of the antigen is further enhanced and the host immune response is fully activated through specific fusion of the exogenous virus envelope protein and GP, meanwhile, the recombinant virus vaccine does not have secondary replication capacity, and the safety is remarkably enhanced.

The invention provides a virus vector mVSV modified by VSV, an mVSV virus vector and a new coronary pneumonia vaccine generated based on mVSV mediation, and the vaccine has better prevention or treatment effect on a new coronary pneumonia virus infected person.

Disclosure of Invention

The invention provides a vector mVSV for developing vaccines for various viruses abused in the present society, a virus vector and a new coronary pneumonia vaccine based on the mVSV virus vector, which is rapidly developed by an mVSV-Vac platform.

mVSV viral vector, vesicular stomatitis virus Indiana strain Indiana VSV gene M encoding amino acids with multiple site modification mutations, the M protein of VSV 51 methionine M replaced phenylalanine F, 110 phenylalanine F replaced alanine A, 225 isoleucine I replaced leucine L, defined as mVSV.

A mVSV virus vector vaccine comprises the mVSV virus vector, wherein a heterologous antigen gene of a target virus is integrated in the mVSV, the heterologous antigen gene is fused or embedded at the N end or the C end of an mVSV envelope GP gene, and the target virus vaccine formed after fusion or embedding of an antigen is defined as an attenuated vesicular stomatitis virus vaccine.

Further, in the case where the heterologous antigen gene is chimeric at the N-terminus or C-terminus of mvv envelope GP gene, the DNA of the heterologous antigen gene is a codon-optimized sequence, and the antigen gene comprises a full-length or partial truncation of spike protein S gene encoding the envelope of the virus of interest.

Further, the full length of the spike protein S gene of the envelope of the virus of interest comprises SEQ ID NO 1 or a gene sequence having an amino acid sequence at least 98% identical to that encoding SEQ ID NO 2, defined as a chimeric antigen gene A; the partial truncation of the spike protein S gene of the target virus envelope comprises the base sequence of SEQ ID NO. 3 or the gene sequence with amino acid having at least 98% identity with the encoding SEQ ID NO. 4, and is defined as a chimeric assembly gene B.

Further, the heterologous antigen gene DNA is integrated into the envelope GP gene coding sequence or into an adjacent non-coding sequence within the mVSV vector gene segment.

Further, in the case that the heterologous antigen gene is fused at the N-terminal or C-terminal of the envelope GP gene, the fusion of the 5 'terminal of the envelope GP gene occurs after the signal peptide of the envelope GP gene, and the fusion of the 3' terminal of the envelope GP gene occurs before the stop codon of the envelope GP gene.

Further, the mvv envelope GP fusion antigenic gene is selected from the RBD segment of the spike protein of the neocoronavirus cove-19 virus, said envelope GP gene being present at any position in the pmVSV-Core backbone vector of the vaccine vector corresponding to the target virus, wherein said envelope GP fusion heterologous antigenic gene comprises the RBD gene or RBD blocker gene encoding the S protein of the target virus, said heterologous antigenic gene comprising the amino acid sequence of SEQ ID NO:5 or encoding SEQ ID NO:6 amino acids of at least 98% identity and is defined as the fused antigen gene C.

A new vaccine for coronary pneumonia based on mVSV mediation, which is characterized in that: the target virus is a new coronary pneumonia virus, the mVSV virus vector is embedded or fused with antigen genes of the COVID-19 virus, the antigen genes are selected from dominant antigen epitopes of spike protein S of the COVID-19 virus pathogen, and the dominant antigen epitopes comprise embedded antigen gene A, embedded assembly gene B or fused antigen gene C.

Furthermore, the antigen gene comprises the full length of the spike protein S gene and the corresponding dominant antigen gene in different S gene truncations, and the dominant antigen gene is selected from the full length gene corresponding to a receptor binding domain RBD for coding the spike protein S or the corresponding truncation gene truncations.

Further, the antigenic genes of the COVID-19 virus are selected from codon optimized synthetic genes encoding one or more receptor binding domains RBD comprising the spike proteins of the human neocorolla pneumovirus, wherein said receptor binding domains RBD comprise the antigenic genes of one or more different mutants of the neocorolla pneumovirus.

A vaccine based on mVSV mediated new coronary pneumonia is prepared by culturing 293T cells in 10cm culture dish, incubating with 293T cells in serum-free DMEM medium for 1-12h with 10-100 ul poxvirus (MOI =1-50), using specific transfection reagents to make five plasmid systems of pmVSV-core-A/B/C (1-15ug), pP (0.1-5ug), pL (0.1-5ug), pN (0.1-5ug) and pVSVG (0.1-5ug) according to specific optimal transfection ratio (optimal ratio needs to be further optimized according to specific application preservation cases), transfecting gene into 293T cells, collecting cell supernatant after 48-72 h, centrifuging at high speed of 25000rpm, further dividing and dissolving in 100ul virus solution, storing at 4 deg.C or-80 deg.C;

1) adopts various ways of intramuscular injection, intravenous injection, nasal drip or oral administration for vaccination

2) Only 1 immunization is needed

3) The immunizing dose is 10⁶-10⁸pfu (mvv-A, B/C).

The technical effects are as follows: compared with the prior art, the invention selects the live virus as a vaccine vector, chimerizes or fuses a specific target antigen gene, utilizes the replication capacity of the vesicular stomatitis virus in cells to efficiently and rapidly express the target antigen, leads T cells to reach the mucosal part given by the vaccine and promotes CD8⁺T cell response and other properties, and specific mucosal immune response can be remarkably enhanced by a specific administration mode (mucosal administration route); compared with VSV in the prior art, the modified VSV (mVSV) provided by the patent of the invention has the advantages of lower toxicity of mVSV virus, higher antigen loading capacity and more stable virus genome. The new coronary pneumonia vaccine related by the invention can cause strong innate immune response due to the specificity of the virus, activate the immune system of the organism, is similar to the function of an adjuvant, can fully discover the target antigen carried by the virus in the process of discovering, identifying and clearing the virus by the immune system, is different from the antigen instability of the common vaccine, and is easy to degrade.

The target antigen of the virus vector can be expressed in cytoplasm in a large amount along with the replication of the virus, and is sufficiently presented to DC cells to cause specific immune response of an organism, and when the mucosal part is adopted to be inoculated with the vaccine, the organism can be induced to generate local acquired mucosal immune response. The mvv-Vac vector system of the present invention is useful for mucosal delivery of prophylactic or therapeutic vaccines for a variety of transmucosal infectious pathogens. The vaccine of the invention can effectively induce the generation of specific mucosal SIgA and systemic IgG aiming at the COVID-19 virus.

The new coronary pneumonia vaccine mediated by the virus vector (mVSV) can be administrated by intramuscular injection, intravenous injection, nasal drip, oral administration and other immunization ways, can solve the defect that the traditional vaccine in the prior art can not induce high-intensity immune response (particularly has low neutralizing antibody titer), makes up the defects that the antigen carried by the traditional vaccine can not effectively stay on the local mucosa, can not be fully presented to immune cells by APC (antigen-associated virus), the activated immune response is weak, the antibody titer is low and the like, meanwhile, the mVSV new corona vaccine related by the invention can be used together with other vector vaccines, the first needle is firstly immunized by the mVSV new corona vaccine, and the second needle is secondarily stimulated by the second virus vector vaccine (adenovirus vector vaccine and poxvirus vector vaccine), so that the acquired immune response aiming at the new corona antigen can be further activated, and the response rate of inoculation is greatly improved.

In conclusion, the mVSV vector mediated novel coronary pneumonia vaccine provided by the invention has the following three characteristics:

1) the core of the vector is a virus vaccine mediated vector, encodes the coronavirus spike protein S and different truncation bodies, and preferably, the antigen protein sequence is mainly derived from a COVID-19 virus strain;

2) packaging the recombinant attenuated vesicular stomatitis virus by a specific modified plasmid (low copy) packaging system, namely a vesicular stomatitis virus recombination subsystem;

3) the immune effect of the mVSV vaccine is obviously improved by optimizing the designed immune path, immune program, immune dose and immune part, the number of neutralizing antibodies of systemic IgG and local sIgA antibodies of mucosa is further enhanced by body induction, and the envelope fused candidate vaccine mVSV-C can further activate the antiviral T cell immunity of the body while inducing specific humoral immune response, form permanent memory and generate the lifelong protection effect.

The mVSV virus vector can be used for researching target virus vaccines besides rampant new coronavirus; furthermore, the recombinant new corona vaccine vector and different immunologic adjuvant compositions with immunological effective quantity can be applied to new corona virus patients, and the curing and preventing effects are obvious; the immune response is the induction of anti-S protein serum antibodies and induces anti-S protein specific protective immune responses that induce specific neutralizing antibody titers in excess of 1 international unit/ml. The present invention provides one or more times of the new crown pneumonia vaccine and adjuvant composition thereof according to clinical manifestations of patients, even vaccine combination of the recombinant new crown pneumonia vaccine or the combined objective viral vector and composition containing adjuvant are provided to the patients within weeks, months or years of the first providing step later, and the patients provided with the new crown vaccine recombinant new crown pneumonia vaccine or adjuvant composition thereof include: the individual exhibits one or more symptoms of a COVID-19 virus, the individual lacks any symptoms of a COVID-19 virus, the individual has been exposed to an individual having a COVID-19 virus, the individual is a child, an elderly person, is exposed to or at risk of a biological weapon, is a member of the military, or is a health care worker.

As is well known in the art of professional technical personnel, the COVID-19 virus has cross reaction with antibodies induced by the antigen S of SARS virus, but the homology of the two is only 78%, and further, the homology of the new crown antigen S gene and the truncation thereof can induce the organism to generate specific antibodies when the identity is lower than 98%, so that the homology of the antigen amino acid of the new crown vaccine developed based on the mVSV vector is not limited by the proportion of 98%, and candidate vaccines based on chimeric or fused specific antigen epitope of the mVSV vaccine vector are all limited by the invention, and the COVID-19 virus is SARS-CoV-2 mentioned in a plurality of articles in China.

Description of the drawings:

FIG. 1 is a schematic diagram of the construction, virus packaging and identification of the pmVSV-Core-A, pmVSV-Core-B plasmid;

FIG. 2 detection of serum-specific antibodies in mice immunized with the mVSV-A, mVSV-B viral vector vaccine;

FIG. 3 construction, viral packaging and characterization of the pmVSV-Core-C plasmid;

FIG. 4 antibody (IgG, IgA) detection in serum following multi-pathway immunization of mVSV-C neocorona vaccine;

FIG. 5 detection of neutralizing antibody levels in mouse sera following multiple routes of administration for mVSV-A, mVSV-B and mVSV-C candidate new corona vaccines;

FIG. 6 is a schematic representation of the packaging of a VSV-spike protein S recombinant virus vaccine by genetic recombinant plasmids;

FIG. 7 relates to toxicity comparisons of different mutant strains and wild strains of VSV, including MTT toxicity test in MEF cells (A), replication of different mutants (B), and safety comparisons of different mutants in mouse models (C).

The following detailed description of the invention will be further described in conjunction with the above drawings

The specific implementation mode is as follows:

the present disclosure will be described in further detail with reference to the following specific examples, which are intended to illustrate but not limit the present invention, and mainly by constructing the protective epitope of the COVID-19 virus on the VSV viral backbone vector (pCore), designing a reverse genetic engineering rapid rescue system for attenuated rhabdovirus VSV by synthetic biology techniques, obtaining a viral vector vaccine capable of stably expressing candidate antigens, obtaining serum after in vivo immunization of mice, and identifying the acquired immune response and the generation of neutralizing antibodies against viruses by biological experiments.

The reagents and consumables adopted by the present disclosure are as follows: q5 Hot start High-Fidelity DNA polymerase (NEB M0493L), Nhe I-HF (NEB R3131L), Xho I (NEB R0146S), T4 DNA library Enzyme (NEBM0202L), E.coli DB3.1 Complex Cells (Takara 9057), TIANGEN small endotoxin-free Medium kit (Tiangen DP118-02), Lipofectamine LTX (Invitrogen 15338100), PBS (HycloneH56.01), DMEM High sugar Medium (Gibco C11995500), diabody (Gibco 15140. sup. -122), fetal bovine Serum (Gibco 10091. sup. 148), Opnin-MEM I Reduced um (Gibco 31985), Milnin-96 well cell culture plates (Corning 3599), Corning cell culture plates (Cor # 3519. sup.) (Cor # 35), Corning-M # 35. sup.) (Cor # 35).

Cell line:

293T and 293T-hACE2 adherent cells were cultured in DMEM high-sugar complete medium at 37 ℃ in a specific culture environment containing 5% CO2 (ThermoBB 150 cell incubator).

Attenuated mvv viral vectors:

in one embodiment, the attenuated mVSV viral vector selects a three-site mutant of the matrix protein (M) of the recombinant vesicular stomatitis virus Indiana strain (gene sequence NC _ 001560.1) having preferably a mutated amino acid position having amino acid substitutions simultaneously at positions 51, 110 and 225: methionine M at position 51 was replaced with phenylalanine F, phenylalanine F at position 110 was replaced with alanine a, and isoleucine I at position 225 was replaced with leucine L.

Example 1 toxicity after three-site mutation of M protein against the Indiana strain of VSV was greatly reduced.

Given that the most main pathogenic gene of VSV wild strain is M, and the M protein can induce apoptosis of host cells, which is the main factor of VSV wild strain infecting artiodactyl animals, the best way to reduce VSV wild strain by synthetic biology technology is to carry out genetic engineering mutation on M gene, and the existing research shows that the neurotoxicity of wild VSV can be reduced after the nonsynonymous mutation of amino acid 51 of M protein, therefore, in the implementation of the technology of the invention, single-site mutation comparison is firstly carried out, methionine is mutated into phenylalanine, alanine, leucine and arginine (control) at 51, as shown in FIG. 7A, partial results can find that mVSV-M51F has weaker cytotoxicity in normal fibriform compared with mVSV-M51R, but M51F still has certain cytotoxicity, and based on this, the method of synthesizing biology mutation on M51F is continued, the 2 mutation M51F-F110 (A/R/L) was obtained, and it was found that in the 2 mutant strain, the toxicity of M51F-F110A was further reduced, but in the subsequent (FIG. 7C) nasal administration of Balb/C mice sensitive to VSV wild strain (simulated nervous system infection) was carried out, and it was found that high dose administration of E9pfu of M51F-F110A resulted in weight reduction of some mice (proportion of 1/4), although a phenomenon of transgression occurred within 5 days after inoculation, but it was still indicated that the attenuation modification of M gene was not optimal, and further, based on the position of the 2 mutation, the present invention found a third mutation site which significantly reduced the toxicity of M protein, that is, amino acid 225, when amino acid 225 was mutated from leucine I to leucine L, the attenuation effect was most significant (partial comparison result is not shown in FIG. 7), further as shown in fig. 7B, at the cellular level, mVSV mutated at three sites of M protein (M51F-F110A-I225L) did not detect any damage to MEF normal fibroblasts (the multiplicity of infection increased to 10, no significant cytopathic effect was observed), different mutants were subjected to challenge experiments in animal models in the same way as shown in fig. 7C, and after 8-week-old mice Balb/C, nasal E9pfu mutant viruses were respectively dripped, and safety preliminary evaluations were performed (intravenous administration was also verified by mouse model administration, the same dose was applied to 6 mice per group, except for the control group, the wild VSV strains produced significant adverse reactions, and mice administered with other mutants showed less symptoms), and statistical results showed that the virus with only 3 mutations did not cause weight loss in mice, and the other mutants all exhibited significant weight loss and then recovery, in addition, after early administration, the VSV wild strain has toxicity reaction of abnormal rise of body temperature, meanwhile, the control group VSV wild strain and the mVSV-M51R have mouse hind leg nerve paralysis, and only the administration mice of the three mutant strains have no adverse reaction, so that the high safety of the attenuated strain to normal mice is proved, no toxic or side effect exists, and no potential neurotoxicity exists.

EXAMPLE 2 construction and identification of two chimeric vaccines based on VSV viral vectors

The S gene sequence of the COVID-19 virus issued by NCBI is optimized by codon so as to be expressed in cells (named as antigen gene A), a plurality of potential antigen epitopes predicted by the COVID-19 virus sequence are combined into a new antigen gene (named as antigen gene B), the antigen gene A and the antigen gene B sequence are respectively synthesized on pCDNA3.1 and pUC57 vectors by Nanjing Kingsry organisms, after the target gene is amplified by PCR, a target band is recovered and purified by a fragment purification kit, the fragment and the VSV pmGFP vector are digested for 3h at 37 ℃ by using restriction endonuclease, MCS1(Xhol) and MCS2(Nhel), the gel is recovered and the target fragment is subjected to ligation reaction and then transferred to a sensitive cell, the construction conditions of positive clone and bacterial liquid are screened by PCR, sequencing is verified and identified (and the two constructed plasmids are respectively named as pmV-Core-A and pm-Core-B), the specific implementation steps are as follows:

1) two chimeric vaccine constructions based on VSV viral vectors were constructed according to (FIG. 1A);

2) primer synthesis and primer information: the primers were synthesized by Suzhou Jinzhi Biotechnology Ltd, wherein the PCR primers and the bacterial solution PCR primers selected for the construction and amplification of the A gene are shown in Table 1:

TABLE 1A Gene amplification and detection primers

3) Wherein, the PCR primers and the bacteria liquid PCR primers selected for amplifying the B gene are shown in the table 2:

TABLE 2B Gene amplification

Obtaining a target gene: PCR amplification of the A gene was carried out using the pCDNA3.1 plasmid with the target gene sequence as a template and the primers in Table 1; the B gene was amplified by PCR using the pUC57 plasmid having the desired gene sequence as a template and the primers shown in Table 2.

The above digestion products were purified according to AxyPrepTM PCR Cleanup kit instructions and the product concentration was determined using Nano-300.

The purified product and the vector were digested simultaneously (37 ℃ for 3 hours).

Electrophoresis was performed using 1% Agarose gel, the correct PCR product was verified using the corresponding DNA marker as a control, the gel band was excised, the remaining PCR product was recovered, and the product concentration was determined using Nano-300.

The purified product was ligated to the vector (16 ℃ overnight at a ligation ratio of 1: 5).

Ligation products were transformed according to the E.coli DB3.1 Competent cells (TaKaRa) instructions.

Single clones on LB (Kana) plates were picked up and cultured in a sterile 1.5mL tube containing 200. mu.L of LB (Kana) medium at 37 ℃ and 250rpm for 3 hours, and then Colony PCR was performed to select positive clones.

After agarose gel electrophoresis identification, positive clones were selected according to the ratio of 1: transferring the mixture at a ratio of 500 into a 15mL shake flask, and performing shake culture at 37 ℃ and 250rpm for 14-16 h.

Plasmid extraction was performed according to the TIANGEN kit instructions for small and medium endotoxin free extraction.

The positive plasmids were identified by double digestion (Xho I and Nhe I at 37 ℃ for 3 h).

After enzyme digestion identification, selecting the plasmid with correct identification for plasmid sequencing.

Packaging the plasmid with correct sequencing by VSV virus according to a standard method, and taking pmVSV-GFP plasmid as a positive packaging control; respectively collecting virus supernatant at 48h and 72h, infecting 293T cells which are pre-paved on a 6-well plate with 300uL of the virus supernatant to be packaged, and respectively naming packaged viruses as mVSV-GFP and mVSV-A, mVSV-B; and (4) collecting cells after cytopathic effect, and detecting the expression level of the antigen by WB.

The plasmid construction results and WB detection results are shown in FIG. 1.

According to the experimental result, after the antigen gene A and the antigen gene B are subjected to PCR amplification, specific bands appear at corresponding positions, and the sizes of the band molecules are correct, which indicates that the target band is successfully amplified (figure 1B); mVSV-GFP positive control fluorescence expression intensity after virus packaging infection is better, and pathological changes also occur after mVSV-A and mVSV-B virus infects cells (figure 1C); western Blot also detected expression of the A and B genes at the corresponding positions (FIG. 1D).

Example 3 the effect of immune responses under different immunization protocols for two chimeric vaccines based on VSV viral vectors.

Detection of specific IgA, IgG antibody levels in mice after different immunization protocols by indirect ELISA: after an ELISA plate is coated by recombinant RBD protein of COVID-19 virus S virus, mouse serum at 21d after being immunized once by three immunization modes of muscle, vein and nasal drip is diluted according to a ratio of 1:200 and then added into a corresponding hole, after incubation for 2h, different types of (IgA, IgG) secondary antibodies are diluted according to a ratio of 1:10000 to detect the level of a specific antibody (figure 2), and the specific operation steps are as follows:

1) diluting the coating antigen (S-RBD) with a coating buffer solution to a final concentration of 5 mu g/ml, taking an enzyme label plate, sequentially adding samples (100 mu l/hole) into the holes, and then placing at 4 ℃ for coating overnight;

2) pouring out the coating solution in the sample hole the next day, washing for 3 times by using a washing buffer solution, and buckling and drying the residual liquid in the sample hole on the filter paper after each washing;

3) then, 200. mu.l of 5% BSA blocking solution was added to each well for blocking, and the wells were left at 37 ℃ for 1h (plates were placed in sealed bags). Pouring off the blocking solution, and washing the sample wells with the washing buffer solution for 1 time;

4) the test sera and the negative sera were mixed in the appropriate ratio (1: 100) diluting, adding into a pore plate, incubating at 37 ℃ for 2h, wherein each pore is 100 ul;

5) pouring out the reaction liquid in the sample hole, washing for 1-3 min by using a washing liquid, washing the plate for 5 times, and draining the residual liquid on the filter paper after each washing;

6) enzyme-labeled secondary antibodies (coat anti-mouse IgG HRP) were diluted in 1:10000 mu.l of the solution is added into each hole after dilution, and then the reaction is carried out for 1h at 37 ℃;

7) pouring out the unbound enzyme-labeled antibody, adding a washing solution for washing for 5 times, wherein the washing is performed for 1-3 min each time, and draining the residual liquid on the filter paper after each washing;

8) adding 100 μ l of freshly prepared color development solution (obtained by mixing solution A and solution B in equal proportion) into each well, standing at room temperature, and reacting for 20min in a dark place;

9) adding 100 mul of ELISA stop solution into each hole to stop reaction;

10) the 96-well plate was placed in a microplate reader and OD450nm was read. Comparing the OD450nm values of the sample to be tested and the negative sample under the same dilution ratio, and determining the positive condition, wherein the OD value of the negative sample is temporarily 2.1 times as that of the positive test standard: OD (positive >2.1 × OD (negative samples).

The results show that when mVSV-A virus, mVSV-B virus and mVSV-GFP virus are immunized in different immunization modes for 21d, the specific IgA and IgG antibody levels in serum rise to significant levels, and the expression levels of the antibodies are greatly different under different immunization routes, wherein the nasal drip method immunization mainly activates IgA mucosal immunity (figure 2A), and veins and muscles mainly cause IgG type immune responses (figure 2B).

Example 4 construction and identification of a fusion vaccine based on a VSV viral vector.

The C fragment was fused to the C-terminus of the VSV-G envelope gene (product number GP-C) on the pVSV-GFP vector, or to the N-terminus of the VSV-G envelope gene (product number (C-GP)) by overlap extension PCR.

After two target genes are respectively amplified by the first round of PCR, a target strip is recovered by using a gel recovery kit, two target fragments of the first round of PCR are respectively taken as templates by the second round of PCR, the upstream primer of the fragment 1 and the downstream primer of the fragment 2 are respectively used for amplification of a fusion fragment, the fragment and a pVSV-GFP vector are subjected to enzyme digestion for 3 hours at 37 ℃ by using restriction endonucleases (MCS 1(MluI) and MCS2 (XhoI)), the gel recovery vector and the target fragment are subjected to ligation reaction and then transferred to a competent cell, a positive clone is screened by a bacterial liquid PCR, and the plasmid construction conditions are verified and identified by enzyme digestion and sequencing, and the specific implementation steps are as follows:

1) the construction of a vesicle fusion type VSV viral vector vaccine was performed according to (FIG. 4A);

2) primer synthesis and primer information: the primers were synthesized by Suzhou Jinzhi Biotechnology Inc., and the specific primer information is shown in the following table:

TABLE 3 two forms of fused C Gene amplification

3) Obtaining a target gene: using pVSV-GFP (amplified VSV-G) with target gene sequence and pCDNA3.1 plasmid containing target gene as template to amplify target segment;

4) carrying out electrophoresis by using 1% Agarose gel, using a corresponding DNA marker as a reference to verify whether a PCR product is correct or not, cutting a gel strip, recovering the residual PCR product, and determining the product concentration by using Nano-300;

5) taking a certain amount of the PCR gel recovered products according to the cloning sequence in the primer table respectively, and carrying out second round of PCR amplification to obtain final fusion envelope gene fragments;

6) carrying out electrophoresis by using 1% Agarose gel, using a corresponding DNA marker as a reference to verify whether a PCR product is correct or not, cutting a gel strip, recovering the rest PCR product, determining the product concentration by using Nano-300, and carrying out double enzyme digestion on a purified product and a vector (Mlul and Xhol, enzyme digestion is carried out for 3 hours at 37 ℃);

7) after enzyme digestion, the enzyme digestion product is purified according to the AxyPrepTM PCR clean kit instruction, and the product concentration is determined by Nano-300;

8) ligating the purified product with a carrier (16 ℃ ligation overnight at a ligation ratio of 1: 5);

9) the ligation products were transformed according to the e.coli DB3.1 competition cells (takara) instructions;

10) selecting a single clone on an LB (Kana) plate, adding the single clone into a sterile 1.5mL tube in which 200 mu L of LB (Kana) culture medium is added in advance, culturing at 37 ℃ and 250rpm for 3h, and then carrying out Colony PCR to screen positive clones;

11) after agarose gel electrophoresis identification, positive clones were selected according to the ratio of 1: transferring 1000 parts of the mixture into a 15mL shake flask, and carrying out shake culture at 37 ℃ and 250rpm for 14-16 h;

12) extracting plasmids according to the instruction of a TIANGEN small-extraction medium-amount kit without endotoxin;

13) the positive plasmids were identified by double digestion (MluI and XhoI at 37 ℃ for 3 h).

14) After enzyme digestion identification, selecting a plasmid with correct identification to perform plasmid sequencing;

15) packaging the plasmid with correct sequencing by VSV virus according to a standard method, and taking pVSV-GFP plasmid as a positive packaging control;

16) the virus supernatants were harvested once at 48h and 72h and 300uL were used to infect a package of 293T cells previously plated in 6-well plates, the viruses were designated mVSV- (GP-C) and mVSV- (C-GP), respectively;

17) and (4) collecting cells after cytopathic effect, and detecting the expression level of the antigen by WB.

The plasmid construction results and WB detection results are shown in FIG. 3.

According to the experimental results, after one round of PCR amplification, specific bands appear at corresponding positions and the sizes of band molecules are correct, and after two rounds of fusion PCR, two fragments are successfully fused together (FIG. 3B); mVSV-GFP positive control fluorescence expression intensity after virus packaging infection is better, and the pathological change condition also appears after mVSV- (GP-C) gene and mVSV- (C-GP) virus infect cells (figure 3C); expression of VSVG was also detected at the corresponding position by Western Blot (fig. 3D).

Example 5 effects of immune response in different immunization protocols for a fusion vaccine based on VSV viral vectors.

Detection of specific IgA, IgG antibody levels in mice after different immunization protocols by indirect ELISA: after an ELISA plate is coated by recombinant RBD protein of COVID-19 virus S virus, the plate is immunized once by three immunization modes of muscle, vein and nasal drip, mouse serum at 21d is diluted according to a ratio of 1:200 and then added into a corresponding hole, after incubation for 2h, different types of (IgA, IgG) secondary antibodies are diluted according to a ratio of 1:10000 to detect the level of a specific antibody (figure 4), and the specific operation steps are as follows:

9) adding 100 mul of ELISA stop solution into each hole to stop reaction;

The results show that when the mVSV- (GP-C) virus, mVSV- (GP-C) virus and mVSV-GFP are immunized for 21d in different immunization modes, the specific IgA and IgG antibody levels in serum rise to significant levels, and the expression levels of the antibodies are greatly different under different immunization routes, wherein the nasal dropping method immunization mainly activates IgA mucosal immunization (figure 4B), and veins and muscles mainly cause IgG type immune responses (figure 4B).

Example 6 immune serum neutralizing antibody detection based on pseudovirus system.

The titer of the generated neutralizing antibody is determined through an in vitro virus neutralization experiment to evaluate the antigen selection difference, high-efficiency protective immunogen is screened out, the titer of the neutralizing antibody which is generated by different groups and has specificity aiming at SARS-CoV-2 is compared, and the optimal vaccine preparation strategy and the optimal vaccination mode are determined, and the specific operation steps are as follows:

1) extinguishing the serum to be detected at 56 ℃ for 30min, centrifuging at 6000g for 3min, and taking the supernatant for later use;

2) 293T-hACE2 cells were passaged and counted and added to 96-well plates (200. mu.L/well containing 8. mu.g/mL polybrene) at 2E4 cells/well;

3) after h, the antibody was serially diluted (1: 2) 10. mu.L/tube with Opti-MEM while a positive control without antibody (20. mu.L of virus solution, final virus concentration 4E5 TU/mL) and a negative control without virus (20. mu.L Opti-MEM) were run;

4) pseudovirus was also serially diluted to 8E5 TU/mL;

5) add 10. mu.L of diluted virus (8E 5 TU/mL) to the 10. mu.L dilution of antibody in step 2 (1: 1 pipetting mix) (final virus concentration 4E5 pfu/mL);

6) incubating in a 5% CO2 incubator at 37 ℃ for 1h, adding into 293T-hACE2 cells, infecting for 24h-48h, and observing fluorescence and pathological changes;

7) taking the dilution multiple of the antibody serum corresponding to the hole with the green fluorescence as the serum neutralization titer;

the results show that mVSV-A, mVSV-B and mVSV-C new corona vaccines have a significant increase in the level of neutralizing antibodies in serum when administered in different modes of administration, compared with the control group, and have a certain difference in the expression level of neutralizing antibodies in different routes of administration, wherein the serum of the mice immunized intravenously induces the highest level of neutralizing antibodies, the intramuscular injection and nasal drip administration have no significant difference, and the mVSV-C group administered intravenously induces the most neutralizing antibodies in three different virus vector vaccines indirectly show that the specific preferred new corona antigen RBD generates neutralizing antibodies against new corona through fusion with VSVG (high immunogenicity) (N-end fusion), indirectly prove that the fusion of the foreign dominant antigen at the N-end of the mVSV vector gene GP can improve the recognition of the antigen in the body by enhancing the envelope display of the dominant antigen in the recombinant virus, inducing the generation of neutralizing antibody and resisting the virus immunological memory.

Further as shown in FIG. 6, the mVSV-mediated COVID-19 virus vaccine series products include mVSV-A/B and mVSV-C, and two different design strategies are disclosed in the present invention, wherein the mVSV-A/B is to integrate the antigen gene of the new coronavirus into the non-enveloped core region of the virus, preferably the chimeric position is at the coding region positions of envelope GP and polymerase L, the virus vector candidate vaccine is a live strain when inoculated because the antigen gene needs to be transcribed and translated into protein by host cells infected in vivo to be subjected to antigen presentation by immune cell DC, the antigen gene can express foreign antigen in vivo along with the replication of the virus to activate the organism to generate specific immune response against the new corona, while the design strategy of another mVSV-C candidate vaccine is quite different as shown in the figure, the technical scheme is to integrate the antigen gene of the new corona virus into the envelope GP gene of the mVSV, preferably, the RBD protein integrated at the N-terminus of the GP gene is expressed by fusion with the GP protein as shown in examples 3 and 4, which demonstrate that the candidate antigenic proteins of mVSV-C-GP vaccine are displayed on the surface of the envelope, so that this type of vaccine can be administered after inactivation (irradiation or high temperature) and is more safe than live attenuated viral vector vaccines, although it is not further described in the examples whether such candidate vaccines can reduce the specific immune response induced by the antigen due to inactivation after inactivation, but the reduction of immunogenicity can improve the response efficiency by increasing the dose of the vaccine upon clinical vaccination, and compensate for the weak immunogenicity due to inactivation.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Ottoming medicine science and technology Co., Ltd, Suzhou

<120> mVSV viral vector, viral vector vaccine thereof and mVSV-mediated novel coronary pneumonia vaccine

<130>2020

<141>2020-03-20

<160>18

<170>SIPOSequenceListing 1.0

<210>1

<211>3822

<212>DNA

<213> Artificial Sequence (Artficial Sequence)

<400>1

atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60

cggacacagc tgccccctgc ctacaccaac agcttcacaa ggggcgtgta ctaccccgac 120

aaggtgttta gatctagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180

aacgtgacct ggttccacgc tatccacgtg tccggcacca acggaacaaa gaggttcgac 240

aacccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300

atcagaggct ggatctttgg aaccacactg gacagcaaga cacagtccct gctgatcgtg 360

aacaacgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa cgatccattc 420

ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt tcgcgtgtac 480

tcctctgcca acaactgtac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540

ggcaagcagg gaaacttcaa gaacctgcgg gagttcgtgt ttaagaacat cgatggctac 600

tttaagatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660

tctgccctgg agccactggt ggatctgccc atcggaatca acatcaccag gtttcagaca 720

ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgct 780

ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840

gagaacggaa ccatcacaga cgctgtggat tgcgccctgg accccctgtc tgagaccaag 900

tgtacactga agagctttac cgtggagaag ggcatctacc agacaagcaa cttccgggtg 960

cagcctaccg agtccatcgt gcgctttccc aacatcacaa acctgtgccc ttttggagag 1020

gtgttcaacg ctacccgctt cgcctccgtg tacgcttgga accggaagcg catctccaac 1080

tgcgtggccg actactctgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140

ggcgtgagcc caacaaagct gaacgacctg tgctttacca acgtgtacgc tgattccttc 1200

gtgatcaggg gagacgaggt gcgccagatc gctcccggcc agacaggaaa gatcgctgac 1260

tacaactaca agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaac 1320

ctggatagca aagtgggcgg aaactacaac tacctgtaca ggctgtttag aaagtctaac 1380

ctgaagccat tcgagcggga catctccaca gagatctacc aggctggctc taccccatgc 1440

aacggagtgg agggcttcaa ctgttacttc cctctgcaga gctacggatt ccagccaaca 1500

aacggcgtgg gataccagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgct 1560

cctgctacag tgtgcggacc aaagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620

ttcaacttta acggactgac cggcacagga gtgctgaccg agtctaacaa gaagttcctg 1680

ccttttcagc agttcggccg ggacatcgcc gataccacag acgctgtgcg cgaccctcag 1740

accctggaga tcctggatat cacaccatgc tccttcggcg gagtgtctgt gatcacacca 1800

ggaaccaaca caagcaacca ggtggccgtg ctgtaccagg acgtgaactg taccgaggtg 1860

cccgtggcta tccacgccga tcagctgacc cctacatgga gggtgtactc taccggcagc 1920

aacgtgttcc agacaagagc cggctgtctg atcggagctg agcacgtgaa caacagctac 1980

gagtgcgaca tccctatcgg cgccggaatc tgtgcttcct accagaccca gacaaactcc 2040

ccaaggagag ccaggtctgt ggctagccag tccatcatcg cctacaccat gagcctgggc 2100

gccgagaact ccgtggctta ctccaacaac tctatcgcta tccctaccaa cttcacaatc 2160

tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220

tacatctgtg gagattccac cgagtgctct aacctgctgc tgcagtacgg ctctttctgt 2280

acccagctga acagagccct gacaggaatc gctgtggagc aggacaagaa cacacaggag 2340

gtgttcgccc aggtgaagca gatctacaag accccaccca tcaaggactt tggcggattc 2400

aactttagcc agatcctgcc cgatcctagc aagccatcca agaggtcttt tatcgaggac 2460

ctgctgttca acaaggtgac cctggctgat gccggcttca tcaagcagta cggcgattgc 2520

ctgggagaca tcgctgccag agacctgatc tgtgcccaga agtttaacgg actgaccgtg 2580

ctgcctccac tgctgacaga tgagatgatc gctcagtaca catctgctct gctggccggc 2640

accatcacaa gcggatggac cttcggcgct ggagctgccc tgcagatccc ctttgccatg 2700

cagatggctt acagattcaa cggcatcgga gtgacccaga acgtgctgta cgagaaccag 2760

aagctgatcg ccaaccagtt taactccgct atcggcaaga tccaggactc tctgagctcc 2820

acagctagcg ccctgggaaa gctgcaggat gtggtgaacc agaacgctca ggccctgaac 2880

accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaacgatatc 2940

ctgagcaggc tggacaaggt ggaggctgag gtgcagatcg acaggctgat cacaggaaga 3000

ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggctgctga gatcagggct 3060

tctgccaacc tggctgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120

gacttttgtg gcaagggata ccacctgatg agcttcccac agtccgcccc tcacggagtg 3180

gtgtttctgc acgtgaccta cgtgccagct caggagaaga acttcaccac agctcccgcc 3240

atctgccacg atggcaaggc ccactttcct cgggagggcg tgttcgtgag caacggaacc 3300

cactggtttg tgacacagcg caacttctac gagccacaga tcatcaccac agacaacaca 3360

ttcgtgtccg gcaactgtga cgtggtcatc ggaatcgtga acaacaccgt gtacgatcct 3420

ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaaccacacc 3480

agccctgacg tggatctggg cgacatctct ggaatcaacg ccagcgtggt gaacatccag 3540

aaggagatcg accggctgaa cgaggtggct aagaacctga acgagtccct gatcgatctg 3600

caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660

atcgccggac tgatcgctat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720

tgttcctgcc tgaagggctg ctgttcttgt ggaagctgct gtaagtttga cgaggacgat 3780

agcgagcctg tgctgaaggg cgtgaagctg cactacacct aa 3822

<210>2

<211>1273

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>2

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro IleAsn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser AlaSer Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu ValLys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr ThrMet Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly LeuThr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg AlaSer Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile

1205 1210 1215

Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile

1220 1225 1230

Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val

1250 1255 1260

Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210>3

<211>447

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

atgttcgtgt ttctggtgct gctgccactg gtgagctccc agtgcaactc taacaatctg 60

gacagcaaag tgggcggcaa ctacaattat ctgtacaggc tgttccgcaa gtccaatctg 120

aagccctttg agcgggatat ctccaccgag atctatcagg ccggctctac accttgcaac 180

ggcgtggagg gcttcaattg ttattttcct ctgcagtctt acggcttcca gccaaccaac 240

ggcgtgggct atcagccata cccaaggaga gccagaggcg gaggaggaag catctacaag 300

acacccccta tcaaggactt tggcggcttc aacttcagcc agatcctggg aggaggagga 360

tccggagccg ccctgcagat ccccttcgcc atgcagatgg cctataggtt taacggcatc 420

ggcgtgaccc agaatgtgct gtactga 447

<210>4

<211>148

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>4

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Asn

1 5 10 15

Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr

20 25 30

Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser

35 40 45

Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly

50 55 60

Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn

65 70 75 80

Gly Val Gly Tyr Gln Pro Tyr Pro Arg Arg Ala Arg Gly Gly Gly Gly

85 90 95

Ser Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe

100 105 110

Ser Gln Ile Leu Gly Gly Gly Gly Ser Gly Ala Ala Leu Gln Ile Pro

115 120 125

Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln

130 135 140

Asn Val Leu Tyr

145

<210>5

<211>708

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>5

atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcaacat cacaaacctg 60

tgcccttttg gagaggtgtt caacgctacc cgcttcgcct ccgtgtacgc ttggaaccgg 120

aagcgcatct ccaactgcgt ggccgactac tctgtgctgt acaacagcgc cagcttcagc 180

accttcaagt gctacggcgt gagcccaaca aagctgaacg acctgtgctt taccaacgtg 240

tacgctgatt ccttcgtgat caggggagac gaggtgcgcc agatcgctcc cggccagaca 300

ggaaagatcg ctgactacaa ctacaagctg cctgacgatt tcaccggctg cgtgatcgcc 360

tggaactcta acaacctgga tagcaaagtg ggcggaaact acaactacct gtacaggctg 420

tttagaaagt ctaacctgaa gccattcgag cgggacatct ccacagagat ctaccaggct 480

ggctctaccc catgcaacgg agtggagggc ttcaactgtt acttccctct gcagagctac 540

ggattccagc caacaaacgg cgtgggatac cagccctacc gcgtggtggt gctgtctttt 600

gagctgctgc acgctcctgc tacagtgtgc ggaccaaaga agagcaccaa cctggtgaag 660

aacaagtgcg tgaacttcaa ctttaacgga ctgaccggca caggataa 708

<210>6

<211>235

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>6

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Asn

1 5 10 15

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

20 25 30

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

35 40 45

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

50 55 60

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

65 70 75 80

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

85 90 95

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

100 105 110

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

115 120 125

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

130 135 140

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

145 150 155 160

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

165 170 175

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

180 185 190

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

195 200 205

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

210 215 220

Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly

225 230 235

<210>7

<211>33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>7

ccgctcgaga tgtttgtttt tcttgtttta ttg 33

<210>8

<211>28

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>8

ctagctagct tatgtgtaat gtaatttg 28

<210>9

<211>27

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>9

ccgctcgaga tgttcgtgtt tctggtg 27

<210>10

<211>30

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>10

ctagctagct cagtggtggt ggtggtggtg 30

<210>11

<211>33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>11

tttacgcgtc actatgaagt gccttttgta ctt 33

<210>12

<211>30

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>12

tgtgatgttc tttccaagtc ggttcatctc 30

<210>13

<211>30

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>13

cttggaaaga acatcacaaa cctgtgccct 30

<210>14

<211>72

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>14

ccgctcgagc gtgatatctg ttagtttttt tcatacctag caggatttga gttatcctgt 60

gccggtcagt cc 72

<210>15

<211>82

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>15

tttacgcgtc actatgaagt gccttttgta cttagccttt ttattcattg gggtgaattg 60

caacatcaca aacctgtgcc ct 82

<210>16

<211>30

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>16

ggtgaacttt cctgtgccgg tcagtccgtt 30

<210>17

<211>29

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>17

ggcacaggaa agttcaccat agtttttcc 29

<210>18

<211>73

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>18

ccgctcgagc gtgatatctg ttagtttttt tcatacctag caggatttga gttactttcc 60

aagtcggttc atc 73

Claims

The mVSV viral vector is characterized by comprising mVSV, wherein the mVSV is obtained after mutation of amino acid of matrix protein M of a vesicular stomatitis virus Indiana strain, and the mutation is carried out on the mutation of 51 th methionine to phenylalanine, 110 th phenylalanine to alanine and 225 th isoleucine to leucine of the matrix protein M.
2. A vaccine comprising the mvv viral vector of claim 1, characterized in that: the gene of the mVSV viral vector is integrated with a heterologous antigen gene of a target virus.
3. The vaccine of claim 2, wherein: the heterologous antigen gene is chimeric or fused in the gene of the mVSV virus vector, and the position of the chimeric or fused is between the envelope G and L genes of the mVSV virus vector.
4. The vaccine of claim 2 or 3, wherein: the target virus is a new coronavirus COVID-19 virus.
5. The vaccine of claim 4, wherein: the antigen gene of the new coronavirus COVID-19 is embedded at the N end or the C end of an envelope GP gene of a mVSV virus vector, the antigen gene of the new coronavirus COVID-19 is a sequence after codon optimization, and the antigen gene of the new coronavirus COVID-19 comprises the full length of a spike protein S gene of the new coronavirus COVID-19 or a partial truncation body comprising an RBD segment gene.
6. The vaccine of claim 5, wherein: the full length of the spike protein S gene of the new coronavirus COVID-19 virus comprises a sequence shown in SEQ ID NO. 1 or a gene sequence of amino acid with at least 98 percent of identity with SEQ ID NO. 2; the partial truncation body of the new coronavirus COVID-19 virus spike protein S gene containing RBD segment gene comprises a sequence shown in SEQ ID NO. 3 or a gene sequence of amino acid with at least 98 percent of identity with SEQ ID NO. 4.
7. The vaccine of claim 4, wherein: the new coronavirus COVID-19 virus antigen gene is embedded to the N end or the C end of the mVSV virus vector envelope GP gene coding sequence.
8. The vaccine of claim 4, wherein: the new coronavirus COVID-19 virus antigen gene is fused at the N end or the C end of an envelope GP gene of an mVSV virus vector, the fusion of the 5 'end of the envelope GP gene is carried out after a signal peptide of the envelope GP gene, and the fusion of the 3' end of the envelope GP gene is carried out before a stop codon of the envelope GP gene.
9. The vaccine of claim 4, wherein: the fused antigen gene of the new coronavirus COVID-19 virus comprises a gene corresponding to an RBD section of a spike protein S of the new coronavirus COVID-19 virus.
10. The vaccine of claim 9, wherein: the fused antigen gene of the new coronavirus COVID-19 comprises a sequence shown in SEQ ID NO. 5 or a gene sequence which codes amino acid with at least 98% of identity with SEQ ID NO. 6.
11. The vaccine of claim 9, wherein: the RBD segment of the spike protein S of the new coronavirus COVID-19 virus is from different mutant strains of the new coronavirus COVID-19 virus.